Crystals of Dispiropyrrolidine Derivatives

ABSTRACT

Crystals of a dispiropyrrolidine compound or a salt thereof which inhibits the action of Mdm2 are provided. The present invention provides crystals of (3′R,4′S,5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxamide or a salt thereof which inhibits Mdm2 and has anti-tumor activity. The present invention also provides a medicament comprising the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/370,160, filed Dec. 6, 2016, which is a continuationapplication of U.S. patent application Ser. No. 15/144,485, filed May 2,2016, now U.S. Pat. No. 9,540,386, issued on Jan. 10, 2107, which is adivisional application of U.S. patent application Ser. No. 14/426,630,filed Mar. 6, 2015, now U.S. Pat. No. 9,359,368, issued on Jun. 7, 2016,which is a national phase application under 35 U.S.C. § 371 ofInternational Patent Application No. PCT/JP2013/073865, filed Sep. 5,2013, which claims priority to Japanese Patent Application No.2012-195761, filed Sep. 6, 2012, the contents of all of which are herebyincorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to crystals of a dispiropyrrolidinecompound having anti-tumor activity by inhibition of murine doubleminute 2 (Mdm2) or a salt thereof.

BACKGROUND ART

p53 is known as an important factor for inhibiting canceration of cells.p53 is a transcription factor that induces the expression of genesinvolved in the cell cycle and cellular apoptosis in response to variousstresses. p53 is thought to inhibit canceration of cells by atranscription regulating function thereof. In fact, deletion or mutationof the p53 gene is observed in about half of human cancer cases.

Meanwhile, overexpression of murine double minute 2 (Mdm2), a type of E3ubiquitin ligase, is known as a factor for canceration of cells that arecancerated in spite of the presence of normal p53. Mdm2 is a protein ofwhich expression is induced by p53. Mdm2 negatively regulates p53 bymediating degradation of p53 by binding to the transcription activitydomain of p53 to decrease the transcription activity of p53, exportingp53 out of the nucleus, and further acting as a ubiquitination ligaseagainst p53. Therefore, it is thought that inactivation of functions ofand degradation of p53 are promoted in cells in which Mdm2 isoverexpressed, resulting in canceration (Non Patent Document 1).

Paying attention to such functions of Mdm2, many approaches have beenattempted using substances that inhibit the suppression of p53 functionsby Mdm2, as candidate anti-tumor agents. Examples of the Mdm2 inhibitorstargeting the Mdm2-p53 binding site have been reported, which includespirooxindole derivatives (Patent Documents 1-15, Non Patent Documents1-3), indole derivatives (Patent Document 16), pyrrolidine-2-carboxamidederivatives (Patent Document 17), pyrrolidinone derivatives (PatentDocument 18) and isoindolinone derivatives (Patent Document 19, NonPatent Document 4).

CITATION LIST Patent Documents

-   Patent Document 1: WO2006/091646-   Patent Document 2: WO2006/136606-   Patent Document 3: WO2007/104664-   Patent Document 4: WO2007/104714-   Patent Document 5: WO2008/034736-   Patent Document 6: WO2008/036168-   Patent Document 7: WO2008/055812-   Patent Document 8: WO2008/141917-   Patent Document 9: WO2008/141975-   Patent Document 10: WO2009/077357-   Patent Document 11: WO2009/080488-   Patent Document 12: WO2010/084097-   Patent Document 13: WO2010/091979-   Patent Document 14: WO2010/094622-   Patent Document 15: WO2010/121995-   Patent Document 16: WO2008/119741-   Patent Document 17: WO2010/031713-   Patent Document 18: WO2010/028862-   Patent Document 19: WO2006/024837

Non Patent Documents

-   Non Patent Document 1: J. Am. Chem. Soc., 2005, 127, 10130-10131-   Non Patent Document 2: J. Med. Chem., 2006, 49, 3432-3435-   Non Patent Document 3: J. Med. Chem., 2009, 52, 7970-7973-   Non Patent Document 4: J. Med. Chem., 2006, 49, 6209-6221

SUMMARY OF INVENTION Technical Problem

A dispiropyrrolidine derivative exhibits excellent Mdm2 inhibitingactivity and is thus expected to be used as a medicament, particularly,as an anticancer agent. In addition, it is of industrially significantimportance to find crystals of the derivative.

Solution to Problem

The present inventors have conducted extensive studies to enhance themedical usefulness of a dispiropyrrolidine derivative that exhibits Mdm2inhibiting activity and has anti-tumor activity and to improve its solidstate properties for this purpose. As a result, the present inventorshave found crystals of a dispiropyrrolidine derivative represented bythe following formula (1) or a salt thereof.

More specifically, the present invention relates to [1] to [14] givenbelow.

[1] A crystal of (3′R, 4′S, 5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″, 2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′, 3″-indole]-5′-carboxamide represented by thefollowing formula (1) or a salt thereof:

[2] A crystal of a compound as defined in [1] having an X-raydiffraction pattern as shown in FIG. 1 as an X-ray powder diffractionpattern obtained by copper Kα radiation (wavelength λ=1.54 angstroms).

[3] A crystal of a compound as defined in [1] having an X-raydiffraction pattern as shown in FIG. 2 as an X-ray powder diffractionpattern obtained by copper Kα radiation (wavelength λ=1.54 angstroms).

[4] A crystal of a compound as defined in [1] having an X-raydiffraction pattern as shown in FIG. 3 as an X-ray powder diffractionpattern obtained by copper Kα radiation (wavelength λ=1.54 angstroms).

[5] A crystal of a hydrochloride of a compound as defined in [1] havingan X-ray diffraction pattern as shown in FIG. 4 as an X-ray powderdiffraction pattern obtained by copper Kα radiation (wavelength λ=1.54angstroms).

[6] A crystal of a methanesulfonate of a compound as defined in [1]having an X-ray diffraction pattern as shown in FIG. 6 as an X-raypowder diffraction pattern obtained by copper Kα radiation (wavelengthλ=1.54 angstroms).

[7] A crystal of an ethanesulfonate of a compound as defined in [1]having an X-ray diffraction pattern as shown in FIG. 7 as an X-raypowder diffraction pattern obtained by copper Kα radiation (wavelengthλ=1.54 angstroms).

[8] A crystal of a benzenesulfonate of a compound as defined in [1]having an X-ray diffraction pattern as shown in FIG. 8 as an X-raypowder diffraction pattern obtained by copper Kα radiation (wavelengthλ=1.54 angstroms).

[9] A crystal of a toluenesulfonate of a compound as defined in [1]having an X-ray diffraction pattern as shown in FIG. 9 as an X-raypowder diffraction pattern obtained by copper Kα radiation (wavelengthλ=1.54 angstroms).

[10] A crystal according to [2] exhibiting characteristic peaks atdiffraction angles 2θ=7.78, 9.14, 10.06, 10.78, 12.18, 13.42, 14.34,15.50, 16.62, 17.06, 17.66, 18.18, 18.74, 20.18, 22.46, 24.90, 25.54,26.94, 27.58, and 28.90 in an X-ray powder diffraction pattern obtainedby copper Kα radiation (wavelength λ=1.54 angstroms).

[11] A crystal according to [3] exhibiting characteristic peaks atdiffraction angles 2θ=7.62, 13.06, 15.10, 17.22, and 21.98 in an X-raypowder diffraction pattern obtained by copper Kα radiation (wavelengthλ=1.54 angstroms).

[12] A crystal according to [4] exhibiting characteristic peaks atdiffraction angles 2θ=9.18, 12.18, 15.58, 16.22, 17.22, 18.42, 18.82,and 19.86 in an X-ray powder diffraction pattern obtained by copper Kαradiation (wavelength λ=1.54 angstroms).

[13] A crystal according to [5] exhibiting characteristic peaks atdiffraction angles 2θ=6.46, 7.86, 9.12, 13.00, 14.42, 19.32, 20.34,20.42, and 21.98 in an X-ray powder diffraction pattern obtained bycopper Kα radiation (wavelength λ=1.54 angstroms).

[14] A crystal according to [6] exhibiting characteristic peaks atdiffraction angles 2θ=7.56, 8.26, 14.00, 16.26, 16.78, 17.72, 18.42,18.62, 20.28, and 23.06 in an X-ray powder diffraction pattern obtainedby copper Kα radiation (wavelength λ=1.54 angstroms).

[15] A crystal according to [7] exhibiting characteristic peaks atdiffraction angles 2θ=6.28, 7.72, 12.62, 14.06, 15.50, 16.62, 16.96,19.68, 21.18, and 25.82 in an X-ray powder diffraction pattern obtainedby copper Kα radiation (wavelength λ=1.54 angstroms).

[16] A crystal according to [8] exhibiting characteristic peaks atdiffraction angles 2θ=6.22, 7.34, 7.90, 12.46, 13.60, 14.22, 15.56,18.86, 19.04, 19.52, 19.72, and 20.54 in an X-ray powder diffractionpattern obtained by copper Kα radiation (wavelength λ=1.54 angstroms).

[17] A crystal according to [9] exhibiting characteristic peaks atdiffraction angles 2θ=6.16, 7.18, 7.88, 12.38, 13.50, 13.88, 15.46,18.46, 19.10, 19.28, 19.66, 20.28, 21.88, and 24.68 in an X-ray powderdiffraction pattern obtained by copper Kα radiation (wavelength λ=1.54angstroms).

[18] A medicament comprising a crystal according to any one of [1] to[17].

Advantageous Effects of Invention

The present invention provides crystals of(3′R,4′S,5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxamideor a salt thereof having Mdm2 inhibiting activity. The crystals of thepresent invention have excellent physicochemical properties in solidstate and are useful as anti-tumor agents.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an X-ray powder diffraction pattern of the crystal (freeform) obtained in Example 1-1. In the drawing, the vertical axisrepresents diffraction intensity indicated in counts/second (cps) units,and the horizontal axis represents diffraction angle indicated in 2θvalues.

FIG. 2 shows an X-ray powder diffraction pattern of the crystal (freeform) obtained in Example 1-2. In the drawing, the vertical axisrepresents diffraction intensity indicated in counts/second (cps) units,and the horizontal axis represents diffraction angle indicated in 2θvalues.

FIG. 3 shows an X-ray powder diffraction pattern of the crystal (freeform) obtained in Example 1-3. In the drawing, the vertical axisrepresents diffraction intensity indicated in counts/second (cps) units,and the horizontal axis represents diffraction angle indicated in 2θvalues.

FIG. 4 shows an X-ray powder diffraction pattern of the crystal(hydrochloride) obtained in Example 2. In the drawing, the vertical axisrepresents diffraction intensity indicated in counts/second (cps) units,and the horizontal axis represents diffraction angle indicated in 2θvalues.

FIG. 5 shows an X-ray powder diffraction pattern of the compound(sulfate) obtained in Example 3. In the drawing, the vertical axisrepresents diffraction intensity indicated in counts/second (cps) units,and the horizontal axis represents diffraction angle indicated in 2θvalues.

FIG. 6 shows an X-ray powder diffraction pattern of the crystal(methanesulfonate) obtained in Example 4. In the drawing, the verticalaxis represents diffraction intensity indicated in counts/second (cps)units, and the horizontal axis represents diffraction angle indicated in2θ values.

FIG. 7 shows an X-ray powder diffraction pattern of the crystal(ethanesulfonate) obtained in Example 5. In the drawing, the verticalaxis represents diffraction intensity indicated in counts/second (cps)units, and the horizontal axis represents diffraction angle indicated in2θ values.

FIG. 8 shows an X-ray powder diffraction pattern of the crystal(benzenesulfonate) obtained in Example 6. In the drawing, the verticalaxis represents diffraction intensity indicated in counts/second (cps)units, and the horizontal axis represents diffraction angle indicated in2θ values.

FIG. 9 shows an X-ray powder diffraction pattern of the crystal(toluenesulfonate) obtained in Example 7. In the drawing, the verticalaxis represents diffraction intensity indicated in counts/second (cps)units, and the horizontal axis represents diffraction angle indicated in2θ values.

FIG. 10 shows adsorption-desorption isotherms of the crystal (free form)obtained in Example 1-1. In the drawing, the vertical axis representschange (%) in the weight of the compound, and the horizontal axisrepresents humidity (% RH).

FIG. 11 shows adsorption-desorption isotherms of the crystal (free form)obtained in Example 1-2. In the drawing, the vertical axis representschange (%) in the weight of the compound, and the horizontal axisrepresents humidity (% RH).

FIG. 12 shows adsorption-desorption isotherms of the crystal(hydrochloride) obtained in Example 2. In the drawing, the vertical axisrepresents change (%) in the weight of the compound, and the horizontalaxis represents humidity (% RH).

FIG. 13 shows adsorption-desorption isotherms of the crystal(methanesulfonate) obtained in Example 4. In the drawing, the verticalaxis represents change (%) in the weight of the compound, and thehorizontal axis represents humidity (% RH).

FIG. 14 shows adsorption-desorption isotherms of the crystal(ethanesulfonate) obtained in Example 5. In the drawing, the verticalaxis represents change (%) in the weight of the compound, and thehorizontal axis represents humidity (% RH).

FIG. 15 shows adsorption-desorption isotherms of the crystal(benzenesulfonate) obtained in Example 6. In the drawing, the verticalaxis represents change (%) in the weight of the compound, and thehorizontal axis represents humidity (% RH).

FIG. 16 shows adsorption-desorption isotherms of the crystal(toluenesulfonate) obtained in Example 7. In the drawing, the verticalaxis represents change (%) in the weight of the compound, and thehorizontal axis represents humidity (% RH).

FIG. 17 is a diagram showing the thermal analysis data of the crystal(free form) obtained in Example 1-1. In the drawing, the vertical axisrepresents temperature difference (DTA) and weight change (TG), and thehorizontal axis represents temperature (° C.). The solid line depicts aDTA curve, and the broken line depicts a TG curve.

FIG. 18 is a diagram showing the thermal analysis data of the crystal(free form) obtained in Example 1-2. In the drawing, the vertical axisrepresents temperature difference (DTA) and weight change (TG), and thehorizontal axis represents temperature (° C.). The solid line depicts aDTA curve, and the broken line depicts a TG curve.

FIG. 19 is a diagram showing the thermal analysis data of the crystal(free form) obtained in Example 1-3. In the drawing, the vertical axisrepresents temperature difference (DTA) and weight change (TG), and thehorizontal axis represents temperature (° C.). The solid line depicts aDTA curve, and the broken line depicts a TG curve.

FIG. 20 is a diagram showing the thermal analysis data of the crystal(hydrochloride) obtained in Example 2. In the drawing, the vertical axisrepresents temperature difference (DTA) and weight change (TG), and thehorizontal axis represents temperature (° C.). The solid line depicts aDTA curve, and the broken line depicts a TG curve.

FIG. 21 is a diagram showing the thermal analysis data of the crystal(methanesulfonate) obtained in Example 4. In the drawing, the verticalaxis represents temperature difference (DTA) and weight change (TG), andthe horizontal axis represents temperature (° C.). The solid linedepicts a DTA curve, and the broken line depicts a TG curve.

FIG. 22 is a diagram showing the thermal analysis data of the crystal(ethanesulfonate) obtained in Example 5. In the drawing, the verticalaxis represents temperature difference (DTA) and weight change (TG), andthe horizontal axis represents temperature (° C.). The solid linedepicts a DTA curve, and the broken line depicts a TG curve.

FIG. 23 is a diagram showing the thermal analysis data of the crystal(benzenesulfonate) obtained in Example 6. In the drawing, the verticalaxis represents temperature difference (DTA) and weight change (TG), andthe horizontal axis represents temperature (° C.). The solid linedepicts a DTA curve, and the broken line depicts a TG curve.

FIG. 24 is a diagram showing the thermal analysis data of the crystal(toluenesulfonate) obtained in Example 7. In the drawing, the verticalaxis represents temperature difference (DTA) and weight change (TG), andthe horizontal axis represents temperature (° C.). The solid linedepicts a DTA curve, and the broken line depicts a TG curve.

DESCRIPTION OF EMBODIMENTS

The present invention relates to crystals of(3′R,4′S,5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxamiderepresented by the following formula (1):

(hereinafter, also referred to as compound (1)) or a salt thereof. Inthis context, the crystals refer to a solid having three-dimensionalregular repeats of atoms (or populations thereof) constituting theinternal structure and are discriminated from amorphous solids, which donot have such a regular internal structure.

Examples of a salt of the compound (1) include any of those described inthe Examples. The compound (1) or the salt thereof may be present in afree or solvate form. The compound (1) or the salt thereof may bepresent in a hydrate form, for example, by absorbing moisture in theair. The solvate is not particularly limited so long as it ispharmaceutically acceptable. Specific examples thereof include ahydrate, an ethanol solvate, and a 2-propanol solvate.

Even crystals derived from the same compound may be generated as aplurality of crystals (crystal polymorphs) differing in internalstructure and physicochemical properties depending on crystallizationconditions. The crystals of the present invention may be any of thesecrystal polymorphs or may be a mixture of two or more crystalpolymorphs.

The crystals of the present invention may have attached water as aresult of absorbing moisture when left in the air, or may form ahydrate, for example, by heating to 25 to 150° C. under ordinaryatmospheric conditions. In addition, the crystals of the presentinvention may also have an attached residual solvent or contain asolvent used in crystallization in their solvate.

In the present specification, the crystals of the present invention maybe defined on the basis of X-ray powder diffraction data. The X-raypowder diffraction can be carried out by measurement and analysisapproaches usually used in the art, and can be performed, for example,by a method described in Examples. In general, hydrates or dehydratesmay vary in their lattice constants through the adsorption or desorptionof crystal water to cause changes in diffraction angle (20) in the X-raypowder diffraction. Also, peak intensity may be changed depending on,for example, difference in crystal growth face or the like (crystalhabit). Accordingly, in the case where the crystals of the presentinvention are defined on the basis of X-ray powder diffraction data,crystals that match therewith in terms of diffraction angles of peaks inthe X-ray powder diffraction and X-ray powder diffraction patterns aswell as hydrates and dehydrates obtained therefrom are also included inthe scope of the present invention.

In a preferred embodiment, a crystal of the present invention is acrystal (free form) having the X-ray powder diffraction pattern shown inFIG. 1 as an X-ray powder diffraction pattern obtained by copper Kαradiation (wavelength λ=1.54 angstroms). Also, the crystal is a crystalexhibiting characteristic peaks at diffraction angles 2θ=7.78, 9.14,10.06, 10.78, 12.18, 13.42, 14.34, 15.50, 16.62, 17.06, 17.66, 18.18,18.74, 20.18, 22.46, 24.90, 25.54, 26.94, 27.58, and 28.90 in an X-raypowder diffraction pattern obtained by copper Kα radiation (wavelengthλ=1.54 angstroms).

In another preferred embodiment, a crystal of the present invention is acrystal (free form) having the X-ray powder diffraction pattern shown inFIG. 2 as an X-ray powder diffraction pattern obtained by copper Kαradiation (wavelength λ=1.54 angstroms). Also, the crystal is a crystalexhibiting characteristic peaks at diffraction angles 28=7.62, 13.06,15.10, 17.22, and 21.98 in an X-ray powder diffraction pattern obtainedby copper Kα radiation (wavelength λ=1.54 angstroms).

In another preferred embodiment, a crystal of the present invention is acrystal (free form) having the X-ray powder diffraction pattern shown inFIG. 3 as an X-ray powder diffraction pattern obtained by copper Kαradiation (wavelength λ=1.54 angstroms). Also, the crystal is a crystalexhibiting characteristic peaks at diffraction angles 28=9.18, 12.18,15.58, 16.22, 17.22, 18.42, 18.82, and 19.86 in an X-ray powderdiffraction pattern obtained by copper Kα radiation (wavelength λ=1.54angstroms).

In another preferred embodiment, a crystal of the present invention is acrystal (hydrochloride) having the X-ray powder diffraction patternshown in FIG. 4 as an X-ray powder diffraction pattern obtained bycopper Kα radiation (wavelength λ=1.54 angstroms). Also, the crystal isa crystal exhibiting characteristic peaks at diffraction angles 28=6.46,7.86, 9.12, 13.00, 14.42, 19.32, 20.34, 20.42, and 21.98 in an X-raypowder diffraction pattern obtained by copper Kα radiation (wavelengthλ=1.54 angstroms).

In another preferred embodiment, a crystal of the present invention is acrystal (methanesulfonate) having the X-ray powder diffraction patternshown in FIG. 6 as an X-ray powder diffraction pattern obtained bycopper Kα radiation (wavelength λ=1.54 angstroms). Also, the crystal isa crystal exhibiting characteristic peaks at diffraction angles 2θ=7.56,8.26, 14.00, 16.26, 16.78, 17.72, 18.42, 18.62, 20.28, and 23.06 in anX-ray powder diffraction pattern obtained by copper Kα radiation(wavelength λ=1.54 angstroms).

In another preferred embodiment, a crystal of the present invention is acrystal (ethanesulfonate) having the X-ray powder diffraction patternshown in FIG. 7 as an X-ray powder diffraction pattern obtained bycopper Kα radiation (wavelength λ=1.54 angstroms). Also, the crystal isa crystal exhibiting characteristic peaks at diffraction angles 28=6.28,7.72, 12.62, 14.06, 15.50, 16.62, 16.96.19.68.21.18, and 25.82 in anX-ray powder diffraction pattern obtained by copper Kα radiation(wavelength λ=1.54 angstroms).

In another preferred embodiment, a crystal of the present invention is acrystal (benzenesulfonate) having the X-ray powder diffraction patternshown in FIG. 8 as an X-ray powder diffraction pattern obtained bycopper Kα radiation (wavelength λ=1.54 angstroms). Also, the crystal isa crystal exhibiting characteristic peaks at diffraction angles 2θ=6.22,7.34, 7.90, 12.46, 13.60, 14.22, 15.56, 18.86, 19.04, 19.52, 19.72, and20.54 in an X-ray powder diffraction pattern obtained by copper Kαradiation (wavelength λ=1.54 angstroms).

In another preferred embodiment, a crystal of the present invention is acrystal (toluenesulfonate) having the X-ray powder diffraction patternshown in FIG. 9 as an X-ray powder diffraction pattern obtained bycopper Kα radiation (wavelength λ=1.54 angstroms). Also, the crystal isa crystal exhibiting characteristic peaks at diffraction angles 2θ=6.16,7.18, 7.88, 12.38, 13.50, 13.88, 15.46, 18.46, 19.10, 19.28, 19.66,20.28, 21.88, and 24.68 in an X-ray powder diffraction pattern obtainedby copper Kα radiation (wavelength λ=1.54 angstroms).

According to another aspect, the present invention relates to amedicament comprising a crystal of the present invention as an activeingredient.

The medicament comprising a crystal of the present invention as anactive ingredient is preferably provided in the form of a pharmaceuticalcomposition comprising a crystal of the present invention and one or twoor more pharmaceutically acceptable carriers. The medicament of thepresent invention is not particularly limited by dosage form and can beadministered orally or parenterally. Preferably, the medicament of thepresent invention is orally administered.

The pharmaceutical composition of the present invention comprises acrystal of the present invention as at least a portion of the compound(I). The pharmaceutical composition may contain a crystal form of thecompound (I) other than the crystals of the present invention. Thecontent of a crystal of the present invention in the pharmaceuticalcomposition can be in the range from 0.01% by weight to 99.9% by weightwith respect to the whole compound (I) in the pharmaceuticalcomposition, for example, 0.01% by weight or larger, 0.05% by weight orlarger, 0.1% by weight or larger, 0.5% by weight or larger, 1% by weightor larger, 2% by weight or larger, 3% by weight or larger, 4% by weightor larger, 5% by weight or larger, 10% by weight or larger, 20% byweight or larger, 30% by weight or larger, 40% by weight or larger, 50%by weight or larger, 60% by weight or larger, 70% by weight or larger,80% by weight or larger, 90% by weight or larger, 95% by weight orlarger, 96% by weight or larger, 97% by weight or larger, 98% by weightor larger, 99% by weight or larger, 99.5% by weight or larger, 99.6% byweight or larger, 99.7% by weight or larger, 99.8% by weight or larger,or 99.9% by weight or larger. The presence or absence of a crystal ofthe present invention in the pharmaceutical composition can be confirmedby an instrumental analysis method (e.g., X-ray powder diffraction,thermal analysis, or infrared absorption spectra) described in thepresent specification.

The crystals of the present invention can be used as an inhibitor ofMdm2 and can be used as a medicament, particularly preferably ananticancer agent, comprising the crystals of the present invention.

In one embodiment of the present invention, a crystal of the presentinvention can be used as a p53-Mdm2 binding inhibitor and/or an Mdm2ubiquitin ligase inhibitor because the compound (1) inhibits the bindingof p53 with Mdm2 and the ubiquitination of p53 by Mdm2.

The condition of the p53-Mdm2 binding can be examined by a methodconventionally used by those skilled in the art to examine bindingconditions between proteins (for example, immunological techniques,surface plasmon resonance techniques, etc.). Examples of methods forexamining the condition of the Mdm2-p53 binding using an immunologicaltechnique include an immuno-sedimentation method andenzyme-linked-immuno-sorbent assay (ELISA). An antibody used in suchimmunological techniques may be an anti-Mdm2 antibody and/or an anti-p53antibody that can directly detect Mdm2 and/or p53. When Mdm2 and/or p53is labeled with a tag (for example, a GST tag or a histidine tag) or thelike, an antibody suitable for labeling (for example, an anti-GSTantibody or an anti-histidine antibody) can be used. Methods forexamining the condition of the Mdm2-p53 binding using an immunologicaltechnique are described in, for example, WO2003/51359, WO2003/51360,U.S. Patent Application Publication No. 2004/259867 or 2004/259884, andWO2005/110996. Methods for examining the condition of the Mdm2-p53binding using a surface plasmon resonance technique are described in,for example, Science, vol. 303, p. 844-848, 2004.

Ubiquitin ligase activity of Mdm2 against p53 can be examined by anubiquitin ligase assay conventionally used by those skilled in the art.The ubiquitin ligase activity can be detected by, for example, comparingubiquitination of p53 by ubiquitin activation enzyme (E1), ubiquitinbinding enzyme (E2), and ubiquitin ligase (E3) (Mdm2) in the presenceand absence of a test compound (for example, refer to WO2001/75145 andWO2003/76608).

In another embodiment, a crystal of the present invention can be used asan inhibitor of suppression of the p53 transcription activity becausethe compound (1) restores functions of p53 as a transcription factorthat is suppressed by Mdm2 by inhibiting the binding of Mdm2 to the p53transcription activation domain. The inhibitor of suppression of the p53transcription activity can be obtained by, for example, measuring themRNA level or the protein level of a protein whose transcription isregulated by p53 (for example, p21^(Waf1/Cip1)) in the presence orabsence of a test compound by an mRNA measuring method (for example,Northern blot) or a protein measuring method (for example, Western blot)conventionally used by those skilled in the art and selecting the testcompound as an inhibitor of suppression of the p53 transcriptionactivity when the mRNA level or the protein level is increased in thepresence of the test compound as compared with that in the absence ofthe test compound. Furthermore, the inhibitor of suppression of the p53transcription activity can also be identified by a reporter assay usingthe reporter activity of a reporter gene including a p53 responsiveelement as an indicator.

In another embodiment, a crystal of the present invention can be used asa p53 degradation inhibitor because the compound (1) inhibitsubiquitination of p53 by Mdm2 and thereby prevents the degradation ofp53 in proteasomes. The p53 degradation inhibitor can be obtained by,for example, measuring the protein level of p53 in the presence orabsence of a test compound by a protein measuring method (for example,Western blot) conventionally used by those skilled in the art andselecting the test compound as a p53 degradation inhibitor when theprotein level is increased in the presence of the test compound ascompared with that in the absence of the test compound.

In another embodiment, a crystal of the present invention can be used asan anti-tumor agent because the compound (1) normalizes functions of p53as a cancer-restraining gene by inhibition of the Mdm2-p53 bindingand/or ubiquitination of p53 by Mdm2.

Cellular growth inhibiting activity can be examined by methods fortesting growth inhibition conventionally used by those skilled in theart. The cell growth inhibition activity can be determined by, forexample, comparing the levels of cellular growth (for example, tumorcells) in the presence or absence of a test compound as described in thefollowing Test Example 2. The levels of cellular growth can be examinedby using, for example, a test system for measuring living cells.Examples of the method for measuring living cells include the[³H]-thymidine uptake test, the BrdU method, the MTT assay, and soforth.

Moreover, in vivo anti-tumor activity can be examined by methods fortesting anti-tumor activity conventionally used by those skilled in theart. The in vivo anti-tumor activity of the present invention can beconfirmed by, for example, transplanting various tumor cells to mice,rats, or the like; after confirming the engraftment of the transplantedcells, orally or intravenously administering the compound of the presentinvention to the animals; a few days or a few weeks later, comparingtumor growth in a drug-non-administered group with that in thecompound-administered group.

A crystal of the present invention can be used for the treatment oftumors or cancers, for example, lung cancer, digestive system cancer,ovary cancer, uterine cancer, breast cancer, prostate cancer, livercancer, head/neck region cancer, blood cancer, renal cancer, skin cancer(malignant melanoma, etc.), retinoblastoma, testicular tumors, andsarcoma, more preferably lung cancer, breast cancer, prostate cancer,colon cancer, acute myeloid leukemia, malignant lymphoma, malignantmelanoma, retinoblastoma, neuroblastoma, and sarcoma. However, thepresent invention is not limited to these cancers.

A medicament of the present invention can contain a crystal of thepresent invention and a pharmaceutically acceptable carrier and can beadministered as various injections such as intravenous injection,intramuscular injection, and subcutaneous injection or by variousmethods such as oral administration or percutaneous administration.Pharmaceutically acceptable carrier means a pharmacologically acceptablematerial (for example, an excipient, a diluent, an additive, a solvent,etc.) that is involved in transport of a composition containing acrystal of the present invention from a given organ to another organ.

A formulation can be prepared by selecting a suitable formulation form(for example, oral formulation or injection) depending on theadministration method and using various conventionally used methods forpreparing a formulation. Examples of oral formulations include tablets,powders, granules, capsules, pills, lozenges, solutions, syrups,elixirs, emulsions, oily or aqueous suspensions, and so forth. In oraladministration, the free compound or a salt form may be used. An aqueousformulation can be prepared by forming an acid adduct with apharmacologically acceptable acid or by forming an alkali metal saltsuch as sodium. As an injection, a stabilizer, a preservative, adissolving aid, and the like can be used in the formulation. Afterfilling a solution that may contain these aids and the like in a vessel,a formulation for use may be prepared as a solid formulation bylyophilization or the like. Furthermore, one dose may be filled in onevessel, or two or more doses may be filled in a vessel.

Examples of solid formulations include tablets, powders, granules,capsules, pills, and lozenges. These solid formulations may containpharmaceutically acceptable additives together with a crystal of thepresent invention. Examples of additives include fillers, extenders,binders, disintegrating agents, dissolution promoting agents, skinwetting agents, and lubricants, and these can be selected and mixed asrequired to prepare a formulation.

Examples of liquid formulations include solutions, syrups, elixirs,emulsions, and suspensions. These liquid formulations may containpharmaceutically acceptable additives together with a crystal of thepresent invention. Examples of additives include suspending agents andemulsifiers, and these are selected and mixed as required to prepare aformulation.

A crystal of the present invention can be used in cancer treatment ofmammals, in particular, humans. The dose and the administration intervalcan be suitably selected depending on the site of the disease, thepatient's height, body weight, sex, or medical history, according to aphysician's judgment. When the compound of the present invention isadministered to a human, the dose range is approx. 0.01 to 500 mg/kgbody weight per day, preferably, approx. 0.1 to 100 mg/kg body weight.Preferably, the compound of the present invention is administered to ahuman once a day, or the dose is divided two to four times, andadministration is repeated at an appropriate interval. Furthermore, thedaily dose may exceed the above-mentioned dose at a physician'sdiscretion, if necessary.

A crystal of the present invention may be used in combination with anadditional anti-tumor agent. Examples thereof include anti-tumorantibiotics, anti-tumor plant constituents, BRMs (biological responsemodifiers), hormones, vitamins, anti-tumor antibodies, molecular targetdrugs, and other anti-tumor agents.

More specifically, examples of alkylating agents include: alkylatingagents such as nitrogen mustard, nitrogen mustard N-oxide, andchlorambucil; aziridine alkylating agents such as carboquone andthiotepa; epoxide alkylating agents such as dibromomannitol anddibromodulcitol; nitrosourea alkylating agents such as carmustine,lomustine, semustine, nimustine hydrochloride, streptozocin,chlorozotocin, and ranimustine; and busulfan, improsulfan tosylate, anddacarbazine.

Examples of various metabolic antagonists include: purine metabolicantagonists such as 6-mercaptopurine, 6-thioguanine, and thioinosine;pyrimidine metabolic antagonists such as fluorouracil, tegafur,tegafur-uracil, carmofur, doxifluridine, broxuridine, cytarabine, andenocitabine; and folic acid metabolic antagonists such as methotrexateand trimetrexate.

Examples of anti-tumor antibiotics include: anti-tumor anthracyclineantibiotics such as mitomycin C, bleomycin, peplomycin, daunorubicin,aclarubicin, doxorubicin, pirarubicin, THP-adriamycin,4′-epidoxorubicin, and epirubicin; and chromomycin A3 and actinomycin D.

Examples of anti-tumor plant constituents include: vinca alkaloids suchas vindesine, vincristine, and vinblastine; taxanes such as paclitaxeland docetaxel; and epipodophyllotoxins such as etoposide and teniposide.

Examples of BRMs include tumor necrosis factors and indomethacin.

Examples of hormones include hydrocortisone, dexamethasone,methylprednisolone, prednisolone, prasterone, betamethasone,triamcinolone, oxymetholone, nandrolone, metenolone, fosfestrol,ethinylestradiol, chlormadinone, and medroxyprogesterone.

Examples of vitamins include vitamin C and vitamin A.

Examples of anti-tumor antibodies and molecular target drugs includetrastuzumab, rituximab, cetuximab, nimotuzumab, denosumab, bevacizumab,infliximab, imatinib mesylate, gefitinib, erlotinib, sunitinib,lapatinib, and sorafenib.

Examples of other anti-tumor agents include cisplatin, carboplatin,oxaliplatin, tamoxifen, camptothecin, ifosfamide, cyclophosphamide,melphalan, L-asparaginase, aceglatone, sizofiran, picibanil,procarbazine, pipobroman, neocarzinostatin, hydroxyurea, ubenimex, andkrestin.

The present invention also includes a method for preventing and/ortreating cancer, comprising administering a crystal of the presentinvention.

The starting material(3′R,4′S,5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxamideor a salt thereof for the crystals of the present invention can beproduced, for example, according to Examples mentioned below.

The X-ray powder diffraction measurement was carried out in Example 1 ata Cu Kα wavelength of λ=1.54 angstroms by a transmission method using D8Discover with GADDS CST (Bruker Axs K.K.) (tube voltage: 40 kV, tubecurrent: 40 mA, scanning range: 2 to 40, scanning rate: 20°/min). TheX-ray powder diffraction measurement was carried out in the otherExamples at a Cu Kα wavelength of λ=1.54 angstroms using areflection-type X-ray powder diffraction apparatus (RINT-TTR III,manufactured by Rigaku Corp.) and a reflection-free sample holder forsamples (tube voltage: 50 kV, tube current: 300 mA, scanning range: 2 to40°, scanning rate: 20°/min, sampling width: 0.020, rotational speed:120 rpm).

The adsorption and desorption measurement apparatuses used were TAinstruments SGA-CX (Examples 2, 6, and 7) and TA instruments VTI-SA(Examples 1, 4, and 5) (temperature: 25° C., humidity: 40, 60, 70, 80,90, 80, 70, 60, 40, 20, 10, 20, 40, 60, 70% RH).

The thermal analysis (TG/DTA) employed TG/DTA6200 manufactured by SIINanotechnology Inc. (rate of temperature rise: 10° C./min, ambient gas:nitrogen, nitrogen gas flow rate: 200 mL/min).

EXAMPLES Example 1

(3′R,4′S,5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxamide

The compound (35 mg, 0.24 mmol) obtained in Step 3 of Reference Example2, triethylamine (0.04 ml, 0.30 mmol), 1-hydroxybenzotriazole (27 mg,0.20 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (46 mg, 0.24 mmol) were added to a N,N-dimethylformamide(4 ml) solution of the compound (100 mg, 0.20 mmol) obtained in Step 3of Reference Example 1 and the resulting mixture was stirred at 50° C.for 1 hour. After cooling, the reaction mixture was diluted with ethylacetate, washed with water, saturated sodium bicarbonate solution, andbrine in this order and then dried over anhydrous sodium sulfate. Thesolvent was evaporated under reduced pressure, then the residue waspurified by NH-silica gel column chromatography[chloroform:methanol=50:1 (v/v)] and the purified product obtained wasdissolved in methanol (10 ml) and stirred at 60° C. for 24 hours. Thesolvent was evaporated under reduced pressure to give 94 mg (76%) of thetitle compound as a solid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.68 (3H, s), 0.95 (3H, s), 1.11-1.27 (2H,m), 1.35-1.81 (8H, m), 2.10-2.17 (1H, m), 2.25-2.32 (1H, m), 3.15 (1H,t, J=10.5 Hz), 3.27 (1H, br s), 3.80 (1H, dd, J=11.0, 2.3 Hz), 3.85-3.95(1H, m), 4.13 (1H, ddd, J=10.8, 4.5, 1.3 Hz), 4.44 (1H, d, J=9.2 Hz),4.64 (1H, d, J=9.2 Hz), 5.46 (1H, d, J=3.7 Hz), 6.49 (1H, d, J=3.7 Hz),6.74 (1H, d, J=1.8 Hz), 7.07 (1H, dd, J=8.2, 1.8 Hz), 7.31 (1H, dd,J=8.2, 2.3 Hz), 7.48-7.52 (2H, m), 7.62 (1H, s), 8.05 (1H, d, J=5.5 Hz).

MS (ESI) m/z: 618 (M+H)⁺.

Example 1-1

A trichloroethylene/ethanol mixture (95/5) (4.75 ml) was added to thecompound (302 mg, 0.49 mmol) obtained in Example 1 and then theresulting mixture was heated to approximately 50° C. for dissolution.The reaction mixture was left standing at room temperature toprecipitate a crystal. The precipitated crystal was collected byfiltration and dried at room temperature to give a crystal. The crystalwas subjected to the measurement of X-ray powder diffraction,simultaneous thermogravimetry and differential thermal analysis(TG/DTA), and adsorption and desorption behaviors.

Alternatively, the crystal can also be obtained using ethyl formate andacetonitrile.

The X-ray powder diffraction pattern is shown in FIG. 1, theadsorption-desorption isotherms are shown in FIG. 10, and the thermalanalysis data (TG/DTA) are shown in FIG. 17.

TABLE 1 Peak of X-ray powder diffraction pattern (relative intensity of22 or more) Peak Relative number 2θ d value intensity 1 7.78 11.35 28 29.14 9.66 72 3 10.06 8.78 22 4 10.78 8.20 39 5 12.18 7.26 67 6 13.426.59 26 7 14.34 6.17 23 8 15.50 5.71 43 9 16.62 5.33 24 10 17.06 5.19 3211 17.66 5.02 28 12 18.18 4.87 77 13 18.74 4.73 100 14 20.18 4.40 72 1522.46 3.95 30 16 24.90 3.57 34 17 25.54 3.48 33 18 26.94 3.31 25 1927.58 3.23 27 20 28.90 3.09 27

Example 1-2

Methanol (3.6 ml) was added to the compound (301 mg, 0.49 mmol) obtainedin Example 1 and then the resulting mixture was heated to approximately50° C. for dissolution. The reaction mixture was left standing at roomtemperature to precipitate a crystal. The precipitated crystal wascollected by filtration and dried at room temperature to give a crystal.The crystal was subjected to the measurement of X-ray powderdiffraction, TG/DTA, and adsorption and desorption behaviors.

Alternatively, the crystal can also be obtained using 2-butanone.

The X-ray powder diffraction pattern is shown in FIG. 2, theadsorption-desorption isotherms are shown in FIG. 11, and the thermalanalysis data (TG/DTA) are shown in FIG. 18.

TABLE 2 Peak of X-ray powder diffraction pattern (relative intensity of14 or more) Peak Relative number 2θ d value intensity 1 7.62 11.59 100 213.06 6.77 14 3 15.10 5.86 47 4 17.22 5.14 27 5 21.98 4.04 19

Example 1-3

Trichloroethylene (1.5 ml) was added to the compound (100 mg, 0.16 mmol)obtained in Example 1 and then the resulting mixture was heated toapproximately 50° C. for dissolution. The reaction mixture was leftstanding at room temperature to precipitate a crystal. The precipitatedcrystal was collected by filtration and dried at room temperature togive a crystal. The crystal was subjected to the measurement of X-raypowder diffraction and TG/DTA.

The X-ray powder diffraction pattern is shown in FIG. 3, and the thermalanalysis data (TG/DTA) are shown in FIG. 19.

TABLE 3 Peak of X-ray powder diffraction pattern (relative intensity of44 or more) Peak Relative number 2θ d value intensity 1 9.18 9.62 100 212.18 7.26 58 3 15.58 5.68 44 4 16.22 5.46 48 5 17.22 5.14 54 6 18.424.81 73 7 18.82 4.71 66 8 19.86 4.47 49

Example 2

A crystal of(3′R,4′S,5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxamidehydrochloride water/2-propanol (IPA) solvate

Concentrated hydrochloric acid (0.026 ml, 0.31 mmol) was added to a2-propanol (2.0 ml) solution of the compound (192 mg, 0.31 mmol)obtained in Example 1 and then the resulting mixture was stirred at roomtemperature for 18 hours. The precipitate was collected by filtration togive 173 mg (85%) of the title crystal.

¹H-NMR (500 MHz, DMSO-d₆) δ: 0.62 (3H, s), 0.92 (3H, s), 1.09-1.58 (6H,m), 1.65-2.07 (5H, m), 2.53-2.94 (1H, m), 3.29-3.73 (5H, m), 4.56-4.76(1H, m), 4.85-5.23 (1H, m), 6.80 (1H, s), 7.01-7.13 (2H, m), 7.14-7.20(1H, m), 7.49-7.74 (2H, m), 8.19-8.42 (1H, m), 8.61-9.08 (1H, m), 10.41(1H, br s), 11.25 (1H, br s).

Anal. Calcd for C₃₀H₃₄Cl₂FN₅O₄.HCl0.75H₂O.IPA: C, 54.48; H, 6.03; N,9.63. Found: C, 54.47; H, 6.14; N, 9.65.

The X-ray powder diffraction pattern of the title crystal is shown inFIG. 4, the adsorption-desorption isotherms thereof are shown in FIG.12, and the thermal analysis data (TG/DTA) thereof are shown in FIG. 20.

TABLE 4 Peak of X-ray powder diffraction pattern (relative intensity of15 or more) Peak Relative number 2θ d value intensity 1 6.46 13.67 35 27.86 11.24 100 3 9.12 9.69 17 4 13.00 6.80 15 5 14.42 6.14 29 6 19.324.59 17 7 20.34 4.36 29 8 20.42 4.35 28 9 21.98 4.04 16

Example 3(3′R,4′S,5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxamidesulfate water/2-propanol (IPA) solvate

Concentrated sulfuric acid (0.005 ml, 0.08 mmol) was added to a2-propanol (0.5 ml) solution of the compound (52 mg, 0.08 mmol) obtainedin Example 1 and then the resulting mixture was stirred at roomtemperature for 2 days. The precipitate was collected by filtration togive 20 mg (34%) of the title compound as a solid.

¹H-NMR (500 MHz, DMSO-d₆) δ: 0.62 (3H, s), 0.92 (3H, s), 1.13-1.61 (6H,m), 1.67-2.09 (5H, m), 2.45-2.88 (1H, m), 3.47-4.01 (5H, m), 4.58-4.77(1H, m), 4.83-5.11 (1H, m), 6.79 (1H, s), 6.98-7.25 (3H, m), 7.51-7.73(2H, m), 8.20-8.41 (1H, m), 8.51-8.73 (1H, m), 8.79-9.05 (1H, m), 10.35(1H, br s), 11.18 (1H, br s).

Anal. Calcd for C₃₀H₃₄Cl₂FN₅O₄.H₂SO₄.0.25H₂O.IPA: C, 49.94; H, 5.71; N,8.82. Found: C, 49.74; H, 5.71; N, 8.85.

The X-ray powder diffraction pattern of the title compound is shown inFIG. 5.

Example 4(3′R,4′S,5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxamidemethanesulfonate hydrate crystal

Methanesulfonic acid (0.026 ml, 0.39 mmol) was added to a 2-propanol (3ml) solution of the compound (221 mg, 0.36 mmol) obtained in Example 1and then the resulting mixture was stirred at room temperature for 16hours. The precipitate was collected by filtration to give 48 mg (19%)of the title crystal.

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.62 (3H, s), 0.92 (3H, s), 1.03-2.01 (11H,m), 2.30 (3H, s), 2.47-2.56 (1H, m), 3.72-3.65 (4H, m), 4.62-4.75 (1H,m), 5.95-5.09 (1H, m), 6.73-6.85 (1H, m), 7.04-7.20 (3H, m), 7.54-7.73(2H, m), 8.23-8.36 (1H, m), 8.60-8.75 (1H, m), 8.83-8.98 (1H, m), 10.83(1H, br s), 11.22 (1H, br s).

Anal. Calcd for C₃₀H₃₄Cl₂FN₅O₄.CH₃SO₃H.2H₂O: C, 49.60; H, 5.64; N, 9.33.Found: C, 49.63; H, 5.45; N, 9.30.

The X-ray powder diffraction pattern of the title crystal is shown inFIG. 6, the adsorption-desorption isotherms thereof are shown in FIG.13, and the thermal analysis data (TG/DTA) thereof are shown in FIG. 21.

TABLE 5 Peak of X-ray powder diffraction pattern (relative intensity of31 or more) Peak Relative number 2θ d value intensity 1 7.56 11.68 48 28.26 10.70 31 3 14.00 6.32 100 4 16.26 5.45 47 5 16.78 5.28 67 6 17.725.00 65 7 18.42 4.81 42 8 18.62 4.76 42 9 20.28 4.38 94 10 23.06 3.85 37

Example 5(3′R,4′S,5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxamideethanesulfonate hydrate crystal

Ethanesulfonic acid (0.032 ml, 0.39 mmol) was added to a 2-propanol (3ml) solution of the compound (221 mg, 0.36 mmol) obtained in Example 1and then the resulting mixture was stirred at room temperature for 23hours. The precipitate was collected by filtration to give 128 mg (49%)of the title crystal.

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.62 (3H, s), 0.92 (3H, s), 1.05 (3H, t,J=7.4 Hz), 1.09-1.59 (6H, m), 1.62-2.06 (5H, m), 2.38 (2H, q, J=7.4 Hz),2.59-3.07 (1H, m), 3.27-3.79 (5H, m), 4.53-4.76 (1H, m), 4.78-5.16 (1H,m), 6.79 (1H, s), 7.00-7.23 (3H, m), 7.51-7.75 (2H, m), 8.21-8.41 (1H,m), 8.48-9.07 (1H, m), 10.35 (1H, br s), 11.19 (1H, br s).

Anal. Calcd for C₃₀H₃₄Cl₂FN₅O₄.C₂H₅SO₃H.4H₂O: C, 48.00; H, 6.04; N,8.75. Found: C, 47.97; H, 5.93; N, 8.56.

The X-ray powder diffraction pattern of the title crystal is shown inFIG. 7, the adsorption-desorption isotherms thereof are shown in FIG.14, and the thermal analysis data (TG/DTA) thereof are shown in FIG. 22.

TABLE 6 Peak of X-ray powder diffraction pattern (relative intensity of23 or more) Peak Relative number 2θ d value intensity 1 6.28 14.06 100 27.72 11.44 39 3 12.62 7.01 39 4 14.06 6.29 36 5 15.50 5.71 23 6 16.625.33 28 7 16.96 5.22 32 8 19.68 4.51 36 9 21.18 4.19 38 10 25.82 3.45 24

Example 6(3′R,4′S,5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxamidebenzenesulfonate hydrate crystal

Benzenesulfonic acid monohydrate (30 mg, 0.17 mmol) was added to a2-propanol (1 ml) solution of the compound (104 mg, 0.17 mmol) obtainedin Example 1 and then the resulting mixture was stirred at roomtemperature for 24 hours. The precipitate was collected by filtration togive 116 mg (89%) of the title crystal.

¹H-NMR (400 MHz, CDCl₃) δ: 0.69 (3H, s), 0.88 (3H, s), 1.09-1.85 (7H,m), 1.88-2.19 (4H, m), 2.53-2.77 (1H, m), 2.95-3.10 (1H, m), 3.53-3.69(1H, m), 3.71-3.89 (2H, m), 4.68-4.85 (1H, m), 5.47-5.80 (2H, m), 6.52(1H, s), 6.77-6.90 (1H, m), 7.03-7.11 (1H, m), 7.24-7.44 (5H, m),7.63-7.98 (4H, m), 8.09-8.43 (1H, m), 10.16 (1H, br s), 10.96 (1H, brs).

Anal. Calcd for C₃₀H₃₄Cl₂FN₅O₄.C₆H₅SO₃H.1.5H₂O: C, 53.80; H, 5.39; N,8.71. Found: C, 53.89; H, 5.40; N, 8.80.

The X-ray powder diffraction pattern of the title crystal is shown inFIG. 8, the adsorption-desorption isotherms thereof are shown in FIG.15, and the thermal analysis data (TG/DTA) thereof are shown in FIG. 23.

TABLE 7 Peak of X-ray powder diffraction pattern (relative intensity of16 or more) Peak Relative number 2θ d value intensity 1 6.22 14.20 25 27.34 12.03 100 3 7.90 11.18 50 4 12.46 7.10 18 5 13.60 6.51 16 6 14.226.22 16 7 15.56 5.69 22 8 18.86 4.70 16 9 19.04 4.66 16 10 19.52 4.54 1911 19.72 4.50 23 12 20.54 4.32 20

Example 7(3′R,4′S,5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxamidep-toluenesulfonate hydrate crystal

An acetonitrile (4 ml) solution of p-toluenesulfonic acid monohydrate(85 mg, 0.45 mmol) was added to an acetonitrile (4 ml) suspension of thecompound (300 mg, 0.50 mmol) obtained in Example 1 and then theresulting mixture was heated at approximately 50° C. for dissolution.The reaction mixture was stirred at room temperature for 1 day. Theprecipitate was collected by filtration to give 255 mg (66%) of thetitle crystal.

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.63 (3H, s), 0.92 (3H, s), 1.09-1.59 (6H,m), 1.66-2.03 (5H, m), 2.29 (3H, s), 2.70-2.91 (1H, m), 3.34-3.74 (5H,m), 4.67 (1H, d, J=10.1 Hz), 4.80-5.11 (1H, m), 6.80 (1H, s), 7.02-7.22(5H, m), 7.43-7.52 (2H, m), 7.55-7.70 (2H, m), 8.23-8.39 (1H, m),8.45-8.74 (1H, m), 10.33 (1H, br s), 11.14 (1H, br s).

Anal. Calcd for C₃₀H₃₄Cl₂FN₅O₄.C₆H₄CH₃SO₃H.1.5H₂O: C, 54.34; H, 5.55; N,8.56. Found: C, 54.06; H, 5.45; N, 8.50.

The X-ray powder diffraction pattern of the title crystal is shown inFIG. 9, the adsorption-desorption isotherms thereof are shown in FIG.16, and the thermal analysis data (TG/DTA) thereof are shown in FIG. 24.

TABLE 8 Peak of X-ray powder diffraction pattern (relative intensity of17 or more) Peak Relative number 2θ d value intensity 1 6.16 14.34 23 27.18 12.30 100 3 7.88 11.21 48 4 12.38 7.14 18 5 13.50 6.55 22 6 13.886.37 23 7 15.46 5.73 25 8 18.46 4.80 21 9 19.10 4.64 27 10 19.28 4.60 1911 19.66 4.51 22 12 20.28 4.38 32 13 21.88 4.06 17 14 24.68 3.60 19

Reference Example 1

[Step 1] (3E/Z)-6-chloro-3-[(2-chloro-3-fluoropyridin-4-yl)methylene]-1,3-dihydro-2H-indol-2-one

N,N-Diisopropylethylamine (0.46 ml, 2.63 mmol) was added to a methanol(130 ml) solution of 6-chloro-1,3-dihydro-2H-indol-2-one (2.20 g, 13.11mmol) and 2-chloro-3-fluoroisonicotinaldehyde (2.20 g, 13.8 mmol) andthe resulting mixture was heated to reflux for 16 hours. After cooling,the precipitate was collected by filtration, washed with cold methanoland dried to give 3.37 g (83%) of the title compound as a solid.

MS (APCI) m/z: 309 (M+H)⁺.

[Step 2] (3′S, 4′R,7′S,8′S,8a′R)-6″-chloro-8′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-3′,4′-diphenyl-3′,4′,8′,8a′-tetrahydro-1′H-dispiro[cyclohexane-1,6′-pyrrolo[2,1-c][1,4]oxazine-7′,3″-indole]-1′,2″ (1″H)-dione

A boron trifluoride-diethyl ether complex (0.15 ml, 1.20 mmol) and 4Amolecular sieves (powder) (3 g) were added to a tetrahydrofuran (30 ml)solution of the compound (1.86 g, 6.00 mmol) obtained in Step 1,(5R,6S)-5,6-diphenylmorpholin-2-one (1.67 g, 6.60 mmol), and4,4-dimethylcyclohexanone (0.83 g, 6.60 mmol) under a nitrogenatmosphere and the resulting mixture was stirred under heating at 70° C.for 7 days. After cooling, insoluble matter was removed by filtrationthrough celite and the filtrate was washed with brine and then driedover anhydrous sodium sulfate. The solvent was evaporated under reducedpressure and the residue was purified by silica gel columnchromatography [n-hexane:ethyl acetate=4:1→1:1 (v/v)] to give 3.39 g(84%) of the title compound as a solid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.21 (3H, s), 0.53 (3H, s), 0.89-1.08 (3H,m), 1.28-1.43 (3H, m), 1.73-1.81 (1H, m), 2.23-2.33 (1H, m), 4.58 (1H,d, J=11.0 Hz), 4.86 (1H, d, J=3.2 Hz), 5.31 (1H, d, J=11.0 Hz), 6.25(1H, d, J=8.3 Hz), 6.67 (1H, dd, J=8.3, 1.8 Hz), 6.72-6.77 (2H, m), 6.93(1H, d, J=1.8 Hz), 7.04-7.17 (6H, m), 7.18-7.25 (3H, m), 7.79 (1H, t,J=4.6 Hz), 7.99 (1H, s), 8.29 (1H, d, J=5.0 Hz).

MS (APCI) m/z: 670 (M+H)⁺.

[Step 3](4′S,5′R)-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxylicacid

The compound (630 mg, 0.94 mmol) obtained in Step 2 was dissolved inacetonitrile (10 ml) and water (4 ml), potassium carbonate (130 mg, 0.94mmol) was added and the resulting mixture was heated to reflux at 85° C.for 16 hours. After cooling, anhydrous magnesium sulfate (113 mg, 0.94mmol) was added and the resulting mixture was stirred at roomtemperature for 15 minutes. After extraction with ethyl acetate, theorganic layer was washed with brine and dried over anhydrous magnesiumsulfate. The solvent was evaporated under reduced pressure to give(4′S,5′R)-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-1′-[(1R,2S)-2-hydroxy-1,2-diphenylethyl]-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxylicacid (650 mg, 100%) as a solid [MS (ESI) m/z: 688 (M+H)]. The carboxylicacid (650 mg, 0.94 mmol) obtained was dissolved in methanol (30 ml) andwater (8 ml), cerium (IV) diammonium nitrate (1.55 g, 2.82 mmol) wasadded under ice cooling and the resulting mixture was stirred at thesame temperature for 30 minutes. Potassium carbonate (780 mg, 5.64 mmol)was added under ice cooling and the resulting mixture was stirred at thesame temperature for 1 hour. Insoluble matter was removed by filtrationthrough celite, then the filtrate was concentrated under reducedpressure and water was added to the residue obtained, followed byextraction with ethyl acetate. The organic layer was washed with brineand dried over anhydrous sodium sulfate. The solvent was evaporatedunder reduced pressure and the residue obtained was purified by silicagel column chromatography [chloroform:methanol=20:1→4:1 (v/v)] to give152 mg (33%) of the title compound as a solid.

¹H-NMR (500 MHz, CD₃OD) δ: 0.74 (3H, s), 0.9 (3H, s), 1.29-1.44 (2H, m),1.48-1.58 (2H, m), 1.64-1.76 (1H, m), 1.94-2.02 (1H, m), 2.11 (1H, ddd,J=14.0, 14.0, 4.0 Hz), 2.43-2.53 (1H, m), 5.07 (1H, d, J=10.3 Hz), 5.32(1H, d, J=10.3 Hz), 6.84 (1H, d, J=1.7 Hz), 7.16 (1H, dd, J=8.3, 2.0Hz), 7.63 (1H, dd, J=8.0, 2.3 Hz), 7.75 (1H, t, J=5.2 Hz), 8.15 (1H, d,J=5.2 Hz).

MS (ESI) m/z: 492 (M+H)⁺.

Reference Example 2

[Step 1] 2,6-anhydro-3,4,5-trideoxy-5-(dibenzylamino)-L-erythro-hexonicacid

Methyl 2,6-anhydro-3,4,5-trideoxy-5-(dibenzylamino)-L-erythro-hexonateMethyl 2,6-anhydro-3,4,5-trideoxy-5-(dibenzylamino)-L-erythro-hexonate(1.60 g, 4.70 mmol) was dissolved in methanol (30 ml), 1N sodiumhydroxide solution (10 ml) was gradually added under ice cooling andthen the resulting mixture was stirred at room temperature for 3 hours.Dowex 50W-X8 was added to the reaction mixture to adjust its pH to 5 to6, insoluble matter was removed by filtration and then the filtrate wasconcentrated under reduced pressure to give 1.7 g (100%) of the titlecompound as a solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.18-1.26 (1H, m), 1.36-1.48 (1H, m),1.79-1.97 (2H, m), 2.62 (1H, t, J=11.0 Hz), 3.18 (1H, t, J=10.4 Hz),3.40 (1H, d, J=11.5 Hz), 3.51-3.61 (4H, m), 3.90-3.99 (1H, m), 7.12-7.38(10H, m).

MS (ESI) m/z: 326 (M+H)⁺.

[Step 2] (2S,5R)-5-(dibenzylamino)tetrahydro-2H-pyran-2-carboxamide

The compound (870 mg, 2.67 mmol) obtained in Step 1 above was dissolvedin N,N-dimethylformamide (30 ml), 1-hydroxybenzotriazole (361 mg, 2.67mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(614 mg, 3.20 mmol) were added and the resulting mixture was stirred atroom temperature for 15 minutes. Ammonium chloride (285 mg, 5.44 mmol)and N,N-diisopropylethylamine (1.86 ml, 10.7 mmol) were added and theresulting mixture was stirred at room temperature for 8 hours. Thereaction mixture was diluted with ethyl acetate and the organic layerwas washed with saturated sodium bicarbonate solution and brine in thisorder and dried over anhydrous sodium sulfate. The solvent wasevaporated under reduced pressure to give 495 mg (57%) of the titlecompound as a solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.35-1.45 (1H, m), 1.60-1.70 (1H, m),2.10-2.18 (1H, m), 2.21-2.28 (1H, m), 2.76 (1H, tt, J=11.4, 4.0 Hz),3.44 (1H, t, J=10.9 Hz), 3.67 (4H, q, J=14.2 Hz), 3.71-3.73 (1H, m),4.04 (1H, dq, J=11.0, 2.1 Hz), 5.35 (1H, s), 6.40 (1H, s), 7.21-7.36(10H, m).

MS (ESI) m/z: 325 (M+H)⁺.

[Step 3] (2S,5R)-5-aminotetrahydro-2H-pyran-2-carboxamide

The compound (490 mg, 1.51 mmol) obtained in Step 2 above was dissolvedin ethanol (10 ml), 20% palladium hydroxide (100 mg) was added and theresulting mixture was stirred at room temperature for 16 hours under ahydrogen atmosphere. The catalyst was removed by filtration throughcelite, then the solvent in the filtrate was evaporated under reducedpressure and the residue was dried to give 215 mg (99%) of the titlecompound as a solid.

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.11-1.22 (1H, m), 1.25-1.35 (1H, m),1.83-1.91 (2H, m), 2.51-2.60 (1H, m), 2.90 (1H, t, J=10.5 Hz), 3.52 (1H,d, J=11.9 Hz), 3.78-3.84 (1H, m), 6.99 (1H, br s), 7.09 (1H, br s).

MS (ESI) m/z: 145 (M+H)⁺.

Test Example 1 Mdm2/p53 Binding Assay

A protein dilution containing 6.25 nM each of His-p53 (fusion protein ofa p53 partial protein having p53 amino acids at positions 1 to 132, witha histidine protein) and GST-Mdm2 (fusion protein of a Mdm2 partialprotein, having Mdm2 amino acids at positions 25 to 108 with leucineresidue 33 substituted by glutamic acid, with glutathione transferase)proteins was prepared using a protein buffer solution (20 mM HEPES pH7.4, 150 mM NaCl, 0.1% BSA). This protein dilution was added in anamount of 8 μL/well to a 384-well plate (384-well low volume NBC,Corning Inc., catalog No: 3676).

Next, a test compound was diluted with DMSO to produce protein buffersolution containing 10% dilution, and this buffer solution was added inan amount of 4 μL/well to the plate.

Subsequently, a solution containing an XL665-labeled anti-His antibody(HTRF monoclonal anti-6HIS antibody labeled with XL665 (catalog No:61HISXLB), Schering/Cisbio Bioassays) and a europium (Eu)-labeledanti-GST antibody (HTRF monoclonal anti-GST antibody labeled witheuropium cryptate, Schering/Cisbio Bioassays, catalog No: 61GSTKLB) atconcentrations of 2.5 μg/mL and 0.325 μg/mL, respectively, was preparedusing an antibody diluting buffer solution (20 mM HEPES pH 7.4, 150 mMNaCl, 0.1% BSA, 0.5 M KF). These dilutions were added in an amount of 8μL/well (total reaction solution volume: 20 μl/well). Then, the platewas left at 25° C. for 1 hour.

Time-resolved fluorescence at 620 and 665 nm was measured at anexcitation wavelength of 320 nm using a plate reader (ARVOsx,PerkinElmer Co., Ltd. or PHERAstar, BMG LABTECH). Ratio (R) wascalculated using the measured values (RFU 620 nm and RFU 665 nm)according to the following formula:

R=(RFU665 nm-BI-C×RFU620 nm)/RFU620 nm

BI: measured value at 665 nm of reaction solution (only each buffersolution) nonsupplemented with each protein, the compound, and theantibodies

C(correction factor)=(A−BI)/D

A and D: each measured value at 665 nm and 620 nm of reaction solutionsupplemented with only Eu-labeled anti-GST antibody solution.

The R value calculated from the well supplemented with His-p53,GST-Mdm2, the test compound, and each antibody was defined as R(sample). The R value calculated from the well supplemented withHis-p53, GST-Mdm2, and each antibody but without the test compound wasdefined as R (control). The R value calculated from the wellsupplemented with GST-Mdm2, the test compound, and each antibody butwithout His-p53 was defined as R (background). T/C was calculated fromthe formula shown below. An IC₅₀ value for Mdm2/p53 binding wascalculated by sigmoid fitting. The results are shown in Table 1.

T/C=(R(sample)−R(background))/(R(control)−R(BACKGROUND))

The compound (1) exhibited an IC₅₀ value of 0.1 μM or lower.

Test Example 2 Cell Growth Inhibition Assay

A cell growth inhibition assay was conducted using human lungcancer-derived cell line NCI-H460 having wild-type p53.

NCI-H460 cells were suspended in a medium (RPMI1640 medium containing10% fetal bovine serum) and the suspension was inoculated in an amountof 500 cells/150 μL/well to a 96-well multiwell plate. A test compoundwas dissolved in DMSO and this solution was diluted with medium toprepare a sample solution (DMSO concentration: 1% or lower). On the nextday of inoculation, medium nonsupplemented with the test compound or thesample solution was added in an amount of 50 μL/well. The MTT assay wasconducted immediately after the medium was added in an amount of 50 μLon the next day of cell inoculation, and after the sample solution orthe medium was added to cells followed by culturing at 37° C. for 3 daysin a 5% CO₂ atmosphere. The MTT assay was conducted as shown below.

A 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, Sigma-Aldrich Co., M-2128) solution was prepared using aphosphate buffer solution (Dulbecco's Phosphate-buffered Saline). ThisMTT solution was added in an amount of 20 μL/well. Then, the plate wascultured at 37° C. for 4 hours in a 5% CO₂ atmosphere. The plate wascentrifuged at 1200 rpm for 5 minutes and then the culture supernatantwas removed by aspiration using a dispenser. DMSO was added in an amountof 150 μL/well to dissolve generated formazan. The plate was stirredusing a plate mixer for uniform color development from each well. Theabsorbance of each well was measured under conditions of OD 540 nm andreference 660 nm using a plate reader (SpectraMax PLUS384, MolecularDevices, CA, USA).

The OD value measured on the day of adding the sample solution wasdefined as S. The OD value measured three days after addition of thesample solution was defined as T. The OD value measured three days afteraddition of the DMSO dilution was defined as C. T/C (%) was determinedat each concentration according to the calculation formula shown belowto prepare a dose response curve, from which 50% growth inhibitionconcentration (GI₅₀ value) was calculated.

T/C(%)=(T−S)/(C−S)×100

The compound (1) exhibited a cell growth inhibiting effect of GI₅₀(μM)<0.1.

(Preparation Example 1)<Capsule>

5 g of a crystal obtained in the Examples, 115 g of lactose, 58 g ofcorn starch, and 2 g of magnesium stearate can be mixed using a V-mixerand then filled in an amount of 180 mg/shell into No. 3 capsule shellsto give capsules.

(Preparation Example 2)<Tablet>

5 g of a crystal obtained in the Examples, 90 g of lactose, 34 g of cornstarch, 20 g of crystalline cellulose, and 1 g of magnesium stearate canbe mixed using a V-mixer and then compressed in a tableting machine in amass of 150 mg/tablet to give tablets.

(Preparation Example 3)<Suspension>

Methylcellulose is dispersed and dissolved in purified water to preparea dispersion medium. A crystal obtained in the Examples is weighed intoa mortar. The dispersion medium is added in small portions to thecrystal while the mixture is well kneaded. Purified water is added toprepare 100 g of suspension.

1. A crystal of(3′R,4′S,5′R)—N-[(3R,6S)-6-carbamoyltetrahydro-2H-pyran-3-yl]-6″-chloro-4′-(2-chloro-3-fluoropyridin-4-yl)-4,4-dimethyl-2″-oxo-1″,2″-dihydrodispiro[cyclohexane-1,2′-pyrrolidine-3′,3″-indole]-5′-carboxamiderepresented by the following formula (1):

wherein: the crystal has the X-ray diffraction pattern as shown in FIG.3 as determined by X-ray powder diffraction obtained by copper Kαradiation (wavelength λ=1.54 angstroms).
 2. The crystal of claim 1,having characteristic peaks at diffraction angles 2θ of 9.18, 12.18,15.58, 16.22, 17.22, 18.42, 18.82, and 19.86 in an X-ray powderdiffraction pattern obtained by copper Kα radiation (wavelength λ=1.54angstroms).
 3. A pharmaceutical composition comprising the crystal ofclaim 1 and a pharmaceutically acceptable carrier.